Antimicrobial resistance and clonality in Acinetobacter baumannii Nemec, A.

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Antimicrobial resistance and clonality in Acinetobacter baumannii

Nemec, A.

Citation

Nemec, A. (2009, September 23). Antimicrobial resistance and clonality in Acinetobacter baumannii. Retrieved from https://hdl.handle.net/1887/14012

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/14012

Note: To cite this publication please use the final published version (if applicable).

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CHAPTER 3 1HPHF$'RO]DQL/%ULVVH6YDQGHQ%URHN3'LMNVKRRUQ/

Diversity of aminoglycoside resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones.

J Med Microbiol 2004; 53: 1233-1240.

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22

Correspondence Alexandr Nemec anemec@szu.cz

Received 26 April 2004 Accepted 18 August 2004

Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones

Alexandr Nemec,

^1,2

Lucilla Dolzani,

³

Sylvain Brisse,

⁴

Peterhans van den Broek

⁵

and Lenie Dijkshoorn

⁵

1National Institute of Public Health, Sˇroba´rova 48, 100 42 Prague 10, Czech Republic

2Department of Medical Microbiology, 3rd Faculty of Medicine, Charles University, Ruska´ 87, 100 00 Prague 10, Czech Republic

3Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Universita di Trieste, I-34127 Trieste, Italy

4Unite´ Biodiversite´ des Bacte´ries Pathoge`nes Emergentes, U 389 INSERM, Institut Pasteur, 75724 Paris Cedex 15, France

5Department of Infectious Diseases, Leiden University Medical Center C5-P, PO Box 9600, 2300 RC Leiden, The Netherlands

The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes and their association with class 1 integrons in three pan- European clones of Acinetobacter baumannii. The study collection included 106 multidrug-resistant strains previously allocated to clone I (n 56), clone II (n 36) and clone III (n 6) and a heterogeneous group of other strains (n 8), using AFLP fingerprinting and ribotyping. The strains were from hospitals of the Czech Republic (n 70; collected 1991–2001) and 12 other European countries (n 36; 1982–1998). Using PCR, at least one of the following aminoglycoside- resistance genes was detected in 101 (95 %) strains: aphA1 (n 76), aacC1 (n 68), aadA1 (n 68), aphA6 (n 55), aadB (n 31), aacC2 (n 7) and aacA4 (n 3). A combination of two to five different resistance genes was observed in 89 strains (84 %), with a total of 12 different combinations. PCR mapping revealed that aacC1, aadA1 and aacA4 were each associated with a class 1 integron, as was the case with aadB for six strains of clone III. Six different class 1 integron variable regions were detected in 78 strains (74 %), with two predominant regions (2.5 and 3.0 kb) in two sets of 34 strains each. The 3.0 kb region contained five gene cassettes (aacC1, orfX, orfX, orfX9, aadA1) and differed from the 2.5 bp region only by one additional orfX cassette. These two integron regions were confined to clones I and II and were found in strains isolated in seven countries between 1982 and 2001. The clone III strains were homogeneous both in resistance genes and in integron variable regions, whereas clones I and II showed a remarkable intraclonal diversity of these properties, with no clear-cut difference between the two clones. Yet, within the Czech clone I and II strains, the diversity of resistance genes and integron structures was limited as compared to those from other countries. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance inA. baumannii.

INTRODUCTION

Acinetobacter baumannii is an important opportunistic pathogen that has the potential to spread among hospitalized patients and persist in the hospital environment (Bergogne- Be´re´zin & Towner, 1996). Recent studies have identiﬁed

Abbreviation: MDR, multidrug-resistant.

The GenBank/EMBL/DDBJ accession number for the sequence of the 3.0 kb integron variable region of NIPH 7 ( CCM 7031 LMG 22454) is AY577724.

Information on properties and origin of the strains used in this study is available as supplementary data in JMM Online.

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23 $PLQRJO\FRVLGHUHVLVWDQFHLQA. baumannii clones

three clones among multidrug resistant (MDR) isolates of A.

baumannii from hospitals in different European countries.

These included clones I and II from north-western Europe in the period 1982–1990 (Dijkshoorn et al., 1996), which were also found to prevail in the Czech Republic between 1991 and 2001 (Nemec et al., 2004), and clone III delineated among western European and Spanish strains from 1997 to 1998 (van Dessel et al., 2004).

Aminoglycosides have long been used for the treatment of infections in hospitalized patients and still are an important alternative for therapy of infections caused by MDR strains.

Previous studies have shown a high diversity of mechanisms of resistance to these antibiotics in the genus Acinetobacter (Shaw et al., 1993; Miller et al., 1995). Resistance to aminoglycosides has been attributed mainly to enzymic inactivation by acetyltransferases, nucleotidyltransferases and phosphotransferases (Shaw et al., 1993), and Acineto- bacter strains often contain multiple enzymes of these classes (Miller et al., 1995; Seward et al., 1998). The genes encoding aminoglycoside-modifying enzymes can be located on plas- mids and transposons (Devaud et al., 1982), and some of these genes have been found on class 1 integrons in MDR A.

baumannii strains in Europe (Seward & Towner, 1999;

Gallego & Towner, 2001; Gombac et al., 2002; Ribera et al., 2004).

Multidrug resistance is a striking feature of the strains belonging to clones I, II and III, and usually includes resistance to aminoglycosides. Overall, there is a great diversity in resistance phenotypes within the clones (Nemec et al., 2004) but the genetic basis of this diversity has not been studied yet. Since multiple mechanisms may give rise to similar phenotypes, it is not known whether there is an association of particular antibiotic-resistance determinants with speciﬁc clones. The aim of the present study was to investigate the genetic basis of aminoglycoside resistance in the pan-European A. baumannii clones. To this aim, the occurrence of different genes encoding aminoglycoside- modifying enzymes and their correlation with aminogly- coside-resistance phenotypes was investigated in a set of well-deﬁned strains from the Czech Republic and other European countries belonging to the three described clones.

In addition, the structural types of class 1 integron variable regions and their association with aminoglycoside-resistance genes were assessed.

METHODS

Bacteria.A total of 106 MDR clinical A. baumannii strains from hospitals in the Czech Republic and other European countries were studied (Table 1). The collection comprised strains classiﬁed into clones I (n 56), II (n 36) and III (n 6), and a heterogeneous group of other strains (n 8). The Czech strains (n 70) were isolated in 23 cities between 1991 and 2001 and were described recently (Nemec et al., 2004). The non-Czech strains (n 36) were isolated in 25 cities from 12 European countries between 1982 and 1998 and, except for four strains

from Eastern Europe, have also been described previously (Dijkshoorn et al., 1996; van Dessel et al., 2004). All Czech strains and 13 other strains of clones I and II (Dijkshoorn et al., 1996) have been characterized uniformly by AFLP, HindIII–HincII ribotyping and biotyping (Nemec et al., 2004). For reasons of harmonization, the remaining strains, i.e. 19 of the study of van Dessel et al. (2004) and four Eastern European strains, were analysed by this panel of methods in the present study.

Antibiotic susceptibility testing.Susceptibility was determined by the disk diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations (NCCLS, 2000) using Mueller–Hinton agar (Oxoid) and the following antimicrobial agents (g per disk): kanamycin (30), gentamicin (10), tobramycin (10), amikacin (30) and netilmicin (30) (Oxoid). MICs of gentamicin, tobramycin, amikacin and netilmicin (MAST Group) were determined by the agar dilution method according to the NCCLS recommendations (NCCLS, 2003).

Detection of aminoglycoside-resistance genes.The presence of genes encoding the following aminoglycoside-modifying enzymes was investigated by PCR: phosphotransferases APH(39)-Ia (aphA1) and APH(39)-VIa (aphA6), acetyltransferases AAC(3)-Ia (aacC1), AAC(3)- IIa (aacC2) and AAC(69)-Ib (aacA4), and nucleotidyltransferases ANT(299)-Ia (aadB) and ANT(399)-Ia (aadA1). The primers were those described by Noppe-Leclercq et al. (1999) for aphA1, aacC1, aacC2, aacA4 and aadB, by Vila et al. (1999) for aphA6 and by Clark et al. (1999) for aadA1. PCR reactions were performed in a ﬁnal volume of 20l containing 10l Taq PCR Master Mix (Qiagen), 0.2 M of each primer and 1.5l of a DNA suspension obtained by alkaline lysis as described by Nemec et al. (2000). The ampliﬁcation reactions were performed in a FTGENE2D thermal cycler (Techne) with the following parameters:

948C for 2 min, followed by 30 cycles of 30 s at 94 8C, 30 s at 55 8C and 60 s at 728C. The presence and sizes of amplicons were assessed by electrophoresis in 2 % agarose gels stained with ethidium bromide.

Integron analysis.The presence of class 1 integrons was determined by PCR amplification of an internal fragment of the integrase gene (intI1) using the primers described by Koeleman et al. (2001). Amplification mixtures and conditions were as specified above. To detect inserted gene cassettes, variable regions of class 1 integrons were amplified with primers 59CS and 39CS, which are complementary to 59 and 39 conserved segments flanking the inserted DNA (Le´vesque et al., 1995). The association of aminoglycoside genes with integrons and the position of the associated genes inside the variable regions were investigated using PCR mapping with primer sets comprising the 59CS primer and a primer for each individual gene (Le´vesque et al., 1995). The amplification protocol used for the 59CS–39CS amplification and PCR mapping consisted of 2 min at 948C, 35 cycles of 45 s at 94 8C, 45 s at 558C and 5 min at 72 8C, and a final extension of 7 min at 72 8C, while the PCR mixtures were prepared as described above. The sequence similarity of amplicons of the same size was investigated by restriction analysis with HinfI and RsaI in separate reactions. The nucleotide sequence of the cloned 3.0 kb variable region from strain NIPH 7 was determined by the dideoxy chain-termination method using an auto- matic DNA sequencer (ALFexpress II; Amersham Biosciences).

RESULTS AND DISCUSSION

Table 1 shows the distribution of the aminoglycoside-

resistance genes, class 1 integron structures and resistance

phenotypes of the strains arranged according to their clonal

types. A more comprehensive table including quantitative

antibiotic susceptibility data is available as supplementary

data in JMM Online.

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&KDSWHU

Table 1.Properties of the 106 strains studied

The strains were allocated to clones on the basis of the grouping obtained by AFLP and HindIII–HincII ribotyping (Nemec et al., 2004) and are classiﬁed successively according to ribotype, resistance gene content, integron type, phenotype and country of isolation. The results of ribotyping and biotyping for NIPH and RUH strains are from Nemec et al. (2004). Resistance phenotypes for kanamycin (K), gentamicin (G), netilmicin (N), tobramycin (T) and amikacin (A) were determined using the disk diffusion test and NCCLS breakpoints for resistance (NCCLS, 2000). RT, HindIII–HincII ribotypes; BT, biotypes according to Bouvet & Grimont (1987);, not found;NG, no growth on minimal medium; BE, Belgium; BG, Bulgaria; CZ, Czech Republic;

DK, Denmark; ES, Spain; FR, France; GR, Greece; HU, Hungary; IT, Italy; NL, the Netherlands; PL, Poland; PT, Portugal.

Clone/strain designation* RT Aminoglycoside-resistance genes†

59CS–39CS amplicon (kb)

Resistance phenotype

BT Country

(years of isolation)

Reference‡

Clone I (n 56) NIPH 188, NIPH 281,

NIPH 307, NIPH 309, NIPH 357, NIPH 1486

1-1 aacC1, aphA1, aphA6, aadB 3.0 K, G, T, A 11 CZ (1993–2001) 1

NIPH 290 1-1 aacC1, aphA1, aphA6, aadB 3.0 K, G, N, T, A 11 CZ (1994) 1

NIPH 1477 1-1 aacC1, aphA1, aphA6, aadB 3.0 K, G, T 11 CZ (2001) 1

LUH 1396 1-1 aacC1, aphA1, aphA6, aadB 2.5 K, G, T, A 6 BG (1997)

LUH 6017 1-1 aacC1, aphA1, aphA6, aadB 2.5 K, G, T, A 6 IT (1998) 3

NIPH 1475, NIPH 1693, NIPH 1729

1-1 aacC1, aphA1, aphA6, aadB 2.5 K, G, T, A 11 CZ (2001) 1

NIPH 409, NIPH 1150, NIPH 1359, NIPH 1488, NIPH 1499, NIPH 1574

1-1 aacC1, aphA1, aphA6 2.5 K, G, A 6 CZ (1996–2001) 1

NIPH 7, NIPH 321, NIPH 392, NIPH 857, NIPH 881, NIPH 1500

NIPH 921 1-1 aacC1, aphA1, aphA6 3.0 K, G, A NG CZ (1997) 1

NIPH 207 1-1 aacC1, aphA1, aphA6 3.0 K, G 11 CZ (1992) 1

RUH 3282 ( GNU 1079) 1-1 aacC1, aphA1, aphA6 3.0 K, G, N, A 11 UK (1990) 1, 2

NIPH 15, NIPH 360 1-1 aacC1, aphA1 3.0 K, G 6 CZ (1991–1994) 1

RUH 436, RUH 510, RUH 2037

1-1 aacC1, aphA1 3.0 K, G 6 NL (1984–1986) 1, 2

RUH 3238 ( GNU 1084), RUH 3239 ( GNU 1083)

1-1 aacC1, aphA1 3.0 K, G 6 UK (1985–1988) 1, 2

NIPH 1587, NIPH 1731 1-1 aacC1, aphA1 3.0 K, G 11 CZ (2001) 1

LUH 6015 1-1 aacC1, aphA1 2.5 K, G 6 IT (1998) 3

NIPH 408 1-1 aacC1, aphA1, aadB 3.0 K, G, T 11 CZ (1996) 1

NIPH 693 1-1 aacC1, aphA6, aadB 3.0 K, G, T, A 11 CZ (1997) 1

NIPH 878 1-1 aacC1, aphA6 2.5 K, G, A 6 CZ (1998) 1

RUH 875 1-1 aphA1, aadB 0.7 K, G, T 6 NL (1984) 1, 2

RUH 3247 ( GNU 1078) 1-1 aphA1, aacA4 0.8 K, G, N, T 6 BE (1990) 1, 2

LUH 5881 1-1 aphA1, aacA4 0.8 K, G, N, T, A 6 ES (1998) 3

NIPH 1358 1-1 aphA6, aadB K, G, T, A 12 CZ (2000) 1

NIPH 470 1-1 aphA6 K, N, A 6 CZ (1997) 1

NIPH 654 1-1 aphA6 K, A 6 CZ (1996) 1

LUH 3584 1-1 aadB K, G, T 6 HU (1995)

NIPH 56 1-1 6 CZ (1992) 1

NIPH 1605 3-1 aacC1, aphA1, aadB 2.5 K, G, T 11 CZ (2001) 1

RUH 3242 ( GNU 1082) 3-1 aacC1, aphA1 3.0 K, G 6 UK (1989) 1, 2

LUH 6125 ( 14C052) 3-1 aacC1, aphA6 3.0 K, G, A 11 PL (1998) 3

NIPH 1722 3-1 aphA6, aadB K, G, T, A 11 CZ (2001) 1

NIPH 10 5-3 aacC1, aphA1, aphA6 3.0 K, G, N, A 6 CZ (1991) 1

Clone II (n 36)

NIPH 455 2-2 aacC1, aphA1, aphA6 2.5 K, G, A 2 CZ (1996) 1

LUH 1398 2-2 aacC1, aphA1, aphA6 2.5 K, G, A 2 BG (1997)

LUH 6021 2-2 aacC1, aphA1, aphA6 2.5 K, G, A 2 PL (1998) 3

(continued overleaf)

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$PLQRJO\FRVLGHUHVLVWDQFHLQA. baumannii clones

Aminoglycoside-resistance genes

A total of 102 strains (96 %) were fully resistant to at least one of kanamycin, gentamicin, tobramycin, netilmicin or ami- kacin, and at least one resistance gene was detected in 101 strains (95 %). The distribution of individual resistance

genes among the strains is shown in Table 2. The observed high frequency of aphA1, aadA1, aacC1, aphA6 and aadB is in agreement with the previously published data on clinical isolates of Acinetobacter spp. (Shaw et al., 1993) and A. baumannii (Seward et al., 1998). There was a good correlation between the content of resistance genes and

Table 1.cont.

Clone/strain designation* RT Aminoglycoside-resistance genes†

59CS–39CS amplicon (kb)

Resistance phenotype

BT Country

(years of isolation)

Reference‡

LUH 1245 2-2 aacC1, aphA1, aadB 2.5 K, G, T 2 HU (1993)

RUH 134 2-2 aacC1, aphA1 3.0 K, G 1 NL (1982) 1, 2

NIPH 24, NIPH 330, NIPH 471, NIPH 499, NIPH 1526, NIPH 1567, NIPH 1696

2-2 aacC1, aphA1 2.5 K, G 2 CZ (1991–2001) 1

NIPH 220 2-2 aacC1, aphA1 2.5 K, G, N, T, A 2 CZ (1993) 1

NIPH 141 2-2 aacC1 2.5 G 2 CZ (1993) 1

LUH 6034 2-2 aphA6, aacC2 K, G, N, A 2 ES (1998) 3

RUH 3422 ( PGS 189) 2-2 aphA1 K 2 DK (1984) 1, 2

NIPH 1462 2-2 aphA1 K 2 CZ (2001) 1

RUH 3245 ( GNU 1080) 2-2 aacC2 G, N 9 UK (1989) 1, 2

LUH 6126 ( 15A250) 2-4 G, N 1 PT (1998) 3

NIPH 1628 2-4 2 CZ (2001) 1

LUH 5868 2-22§ aphA1 K, G, N, T, A 2 FR (1997) 3

RUH 3240 4-2 aacC1, aacC2 2.5 G, N 2 UK (1989) 1, 2

NIPH 1469 4-2 aphA6 K, A 2 CZ (2001) 1

NIPH 1362, NIPH 1711 4-2 2 CZ (2000–2001) 1

LUH 6011 4-21§ aphA1, aacA4 2.2 K, N, T 1 GR (1997) 3

NIPH 657, NIPH 720, NIPH 732, NIPH 1523

LUH 5865 26-2§ aacC2 G, N 2 ES (1998) 3

LUH 6024 26-2§ aphA6, aacC2 K, G, N, A 2 ES (1998) 3

LUH 6044, LUH 6029 26-2§ aphA6, aacC2 K, G, N, T, A 2 ES (1998) 3

Clone III (n 6) LUH 6028, LUH 6037,

LUH 6035

25-1 aphA6, aadB 0.75 K, G, T, A 9 ES (1997–1998) 3

LUH 6009, LUH 5874 25-1 aphA6, aadB 0.75 K, G, T, A 9 FR (1997) 3

LUH 5875 25-1 aphA6, aadB 0.75 K, G, T, A 9 NL (1997) 3

Other strains (n 8)

NIPH 1717 2-6 aphA1 K 6 CZ (2001) 1

NIPH 301 2-7 aphA1 K, G, N 6 CZ (1994) 1

NIPH 47 8-1 aphA1, aadB K, G, T 6 CZ (1991) 1

NIPH 1445 21-16 aphA1, aadB K, G, T 9 CZ (2000) 1

NIPH 335 21-16 aadB K, G, T 9 CZ (1994) 1

NIPH 1497 23-19 aphA1 K, G, N, T 6 CZ (2001) 1

NIPH 1683 23-19 aphA1 K, G 6 CZ (2001) 1

NIPH 1734 24-20 aphA6, aadB K, G, N, T, A New CZ (2001) 1

*The strain designations used in the previous studies are in parentheses.

†The aacC1 gene was always found in association with aadA1. Genes integrated in class 1 integrons (as concluded from the results of PCR mapping) are underlined.

‡1, Nemec et al. (2004); 2, Dijkshoorn et al. (1996); 3, van Dessel et al. (2004).

§Novel ribotypes found for clone II strains. These ribotypes differed only in one or two band positions from those typical for clone II.

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26 &KDSWHU

resistance phenotypes (Table 1). Strains with aphA1, aacC1 or aphA6 were found to be resistant to kanamycin, genta- micin or kanamycin+amikacin, respectively, while aadB was associated with the resistance to kanamycin, gentamicin and tobramycin. In some strains, the presence of a resistance gene was associated only with intermediate or decreased suscept- ibility to a given antibiotic, e.g. the MICs for tobramycin in ﬁve strains with aacC2 were in the range 4–8 g ml

¹

. High- level resistance to netilmicin was predominantly associated with the genes encoding netilmicin-modifying enzymes (i.e.

10 out of 16 strains with MICs >64 g ml

¹

carried either aacA4 or aacC2) while the majority of strains negative for these genes (71 %) showed intermediate or decreased sus- ceptibility to this antibiotic (MICs 4–16 g ml

¹

). Low-level resistance to netilmicin, as frequently found in Acinetobacter spp., does not seem to be linked to enzymic modiﬁcation (Miller et al., 1995) but might be associated with the AdeABC efﬂux pump recently described in A. baumannii (Magnet et al., 2001). In several strains with high-level aminoglycoside resistance (e.g. LUH 5868, NIPH 301 or NIPH 1497), none of the genes possibly involved in this resistance was found, which is also suggestive of additional resistance mechanisms.

Eighty-nine strains (84 %) had a combination of two to ﬁve different resistance genes, and a total of 12 different combinations were encountered (Table 1). The most fre- quent combinations, aadA1 + aacC1 + aphA1 (n 24) and aadA1 + aacC1 + aphA1 + aphA6 (n 23), were detected in both clones I and II, while the combination of aadA1 + aac- C1 + aphA1 + aphA6 + aadB (n 13) was conﬁned to clone I. Combinations of two or three genes encoding resistance to

the same antibiotic (i.e. gentamicin or kanamycin) were found in 56 strains (53 %). The genes (aphA1, aphA6, aadB and aacA4) encoding kanamycin resistance can serve as an example. In contrast to the other genes, aphA1 encodes resistance to kanamycin but not to other clinically important aminoglycosides such as gentamicin, amikacin or netilmicin.

Forty-ﬁve out of the 97 strains resistant to kanamycin carried both aphA1 and at least one of the other kanamycin- resistance genes, 31 strains only contained aphA1 and 21 strains carried kanamycin-resistance genes other than aphA1.

Thirteen out of 30 Czech kanamycin-resistant strains from 2000 to 2001 contained both aphA1 and another kanamycin- resistance gene while other 13 strains carried aphA1 alone.

This, together with a decrease in kanamycin prescribing in the early 1990s, may indicate the stability of the aphA1 gene in the A. baumannii population in the absence of an apparent selective advantage conferred by this gene.

Structural types of the variable regions of class 1 integrons and their association with

aminoglycoside-resistance genes

All strains were investigated for the presence of the integrase gene intI1 and variable regions of class 1 integrons. Seventy- eight strains (74 %) gave a positive reaction for the intI1 gene and using the 5 9CS and 39CS primers, each of these strains yielded a single PCR product of between 0.7 and 3.0 kb. In total, amplicons of six different sizes were detected (Table 3).

Amplicons of the same size gave identical restrictions patterns with HinfI or RsaI, which is indicative of their structural homogeneity. Comparison of the restriction patterns did not suggest a structural similarity between

Table 2.Distribution of aminoglycoside-resistance genes and class 1 integron variable regions among the A. baumannii strains classiﬁed into clonal and geographical groups

Resistance gene/

variable region

Clone I Clone II Clone III

(n 6)

Other Czech strains (n 8)

Total (n 106) Czech

(n 41)

Non-Czech (n 15)

Czech (n 21)

Non-Czech (n 15) Resistance gene

aacC1 36 11 16 5 68 (64 %)

aadA1 36 11 16 5 68 (64 %)

aphA1 34 13 16 7 6 76 (72 %)

aphA6 32 4 6 6 6 1 55 (52 %)

aadB 16 4 1 6 4 31 (29 %)

aacC2 7 7 (7 %)

aacA4 2 1 3 (3 %)

Variable region (kb)

3.0 23 8 2 1 34 (32 %)

2.5 13 3 14 4 34 (32 %)

2.2 1 1 (1 %)

0.8 2 2 (2 %)

0.75 6 6 (6 %)

0.7 1 1 (1 %)

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$PLQRJO\FRVLGHUHVLVWDQFHLQA. baumannii clones

different amplicons, except for the 2.5 and 3.0 kb amplicons, the HinfI patterns of which differed only by an additional 400 bp fragment present in the 3.0 kb amplicon.

Possible association of integrons with aminoglycoside-resis- tance genes was investigated by PCR mapping of all integron- positive strains with the 5 9CS primer in conjunction with primers from aacC1, aacC2, aacA4, aadB or aadA1. The results revealed the association of the 3.0 and 2.5 kb variable regions with aacC1 and aadA1, the 2.2 and 0.8 kb regions with aacA4, and the 0.75 kb region with aadB (Table 3). The aacC1, aadA1 and aacA4 genes were found exclusively as part of integrons while aadB was integron-associated only in the strains of clone III. Taken together with the ampliﬁed external non-coding regions, the known sizes of the aacA4 and aadB cassettes (e.g. accession nos AJ313334 and AF221902, respectively) were sufﬁcient to account for the entire regions of the 0.8 and 0.75 kb amplicons, respectively.

The results of PCR mapping indicated that the aacC1 cassette was located at the 5 9 end of both the 2.5 and 3.0 kb variable regions while aadA1 was located at the 3 9 end of both of these regions. In the case of the 2.2 kb variable region, the aacA4 gene was found at the 5 9 end of the region.

Gombac et al. (2002) characterized a variable region of 2.5 kb of class 1 integrons found in Italian strains of A. baumannii.

This region consisted of four cassettes, i.e. aacC1, two open reading frames of unknown functions (orfX and orfX 9) and aadA1 (accession no. AJ310480). The restriction patterns of the 2.5 kb amplicons of the present study appeared to be identical to those of Gombac et al. (2002) (not shown).

Sequencing of the 3.0 kb amplicon obtained from isolate NIPH 7 revealed an array of cassettes with the order aacC1, orfX, orfX, orfX 9, aadA1, with both orfXs having the same sequence. Thus the 2.5 and 3.0 kb variable regions differ only by one orfX cassette. To our knowledge, this array of gene cassettes has been found only in a Serratia marcescens strain as part of a class 1 integron associated with a Tn1696-like transposon (Centro´n & Roy, 2002; accession no. AF453999), where the orfX and orfX 9 cassettes were termed orfP and orfQ, respectively.

Dissemination and stability of the class 1 integron structures

The structurally related 2.5 and 3.0 kb variable regions were by far the most prevalent integron structures of the present study (Table 2). They were found in both clones I and II but not in the other strains. The 3.0 kb region was detected in 34 strains isolated in four countries between 1982 and 2001 while the 2.5 kb region was found in 34 strains obtained from six countries between 1989 and 2001 (Table 1), indicating the spread of these structures over a relatively large time period.

In addition, the comparison of the sizes, restriction patterns and gene cassette contents of the amplicons of 0.75, 0.8 and 2.2 kb with those of other studies (Gallego & Towner, 2001;

Gombac et al., 2002; Ribera et al., 2004) suggested the geographical dissemination of other class 1 integrons with structurally related variable regions. It has been proposed that class I integrons comprise conserved and stable variable regions, with resistance genes transferred more often as part of the entire integron structure than as individual cassettes (Martinez-Freijo et al., 1999). Indeed, the complexity of the 2.5 and 3.0 kb regions, their distinctness from other variable regions found in A. baumannii and their wide dissemination are indicative of the relative stability of these structures.

Intraclonal and geographical diversity of aminoglycoside-resistance genes and integrons Table 2 shows the distribution of the resistance genes and integron structures among the strains classified into clonal and Czech or non-Czech groups. The clone III strains were homogeneous in all properties, which suggests a recent expansion from a common ancestor. In contrast, the strains of clones I and II showed a remarkable diversity of both resistance genes and integron variable regions. As many as nine and seven resistance gene combinations (including two to five different genes) were observed among the strains of clones I and II, respectively (Table 1). This is consistent with their intraclonal variability in biotype, serotype and plasmid profile and provides further evidence that these clones are relatively old groups that have been undergoing diversifica- tion (Nemec et al., 2004). Thus intraclonal clusters of isolates with identical or highly similar genomic markers and content of resistance genes may represent particular strains (or

Table 3.Characteristics of the variable regions of class 1 integrons found in this study

Variable region (kb) HinfI digestion products (bp)* Inserted gene cassettes detected by PCR mapping

3.0 660, 490, 460, 400, 330, 250, 210, 110, 40 aacC1, aadA1

2.5 660, 490, 460, 330, 250, 210, 110, 40 aacC1, aadA1

2.2 770, 690, 430, 160 aacA4

0.8 500, 160 aacA4

0.75 350, 220, 190 aadB

0.7 470, 260

*Fragment sizes as determined by agarose gel analysis; double fragments (as derived from sequence analysis) are underlined.

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&KDSWHU

subclones) that emerge in a restricted geographical area. For example, all four Czech strains of ribotype R6-4 had the same resistance genes (aadA1, aacC1, aphA1, aphA6) while the aacC2 gene was found in all Spanish isolates of ribotype 26-2 (Table 1).

The strains of clones I and II from the Czech Republic showed a limited spectrum of both resistance genes and integron structures as compared to those from other countries (Table 1). The Czech strains contained only the 2.5 and 3.0 kb integron structures and no aacA4 or aacC2 genes. In contrast, the aacA4 and aacC2 genes and class I integrons with regions other than those of 2.5 and 3.0 kb were found in clones I and II strains from Western Europe. The variations between geographically separated populations of clonally related strains may reﬂect local differences in composition of pools of resistance genes and/or differences in antibiotic usage (Miller et al., 1995). Interestingly, Czech populations of both clones I and II shared all resistance genes (except for aadB found in clone I only) and integron variable regions. A possible explanation is that the two highly prevalent clonal groups of A. baumannii co-occupying a particular ecological niche and geographical region may more readily share genetic pools via horizontal gene transfer.

In conclusion, our study results show a remarkable intraclo- nal diversity of genes encoding aminoglycoside-modifying enzymes in the pan-European clones I and II. Identical resistance genes, gene combinations and class 1 integrons associated with these genes were found in both clones, indicating that horizontal gene transfer plays an important role in the dissemination of aminoglycoside resistance. In addition, the uniformity of the properties found in clusters of isolates within these clones suggests that clonal expansion further facilitates the spread of resistance. To unravel whether there are properties responsible for the wide occurrence of clones I and II still remains a challenging task.

It is conceivable that both the ancient acquisition of some resistance determinants and the high capacity to develop or acquire resistance have contributed to their success. The investigation of mechanisms of resistance to other groups of antibiotics and molecular characterization of mobile struc- tures carrying resistance genes in taxonomically and epide- miologically well-deﬁned strains will contribute to better a understanding of the evolution of multidrug resistance in A.

baumannii.

ACKNOWLEDGEMENTS

Part of this work was presented as poster P742 at the 13th European Congress of Clinical Microbiology and Infectious Diseases, Glasgow, UK, 2003. We thank R. Bressan, M. Maixnerova´ and T. J. K. van den Reijden for excellent technical assistance. We also thank Dr L. Kiss (Debrecen, Hungary) and Dr E. Savov (Soﬁa, Bulgaria) for generous provision of strains. This study was supported by research grant no. 310/

01/1540 of the Grant Agency of the Czech Republic.

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